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The Na,K-ATPase α 2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca 2+ , whereas α 1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α 2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α 2 is preferentially expressed with β 2 in T-tubules of cardiac myocytes, forming α 2 β 2 heterodimers. We have expressed human α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 in Pichia pastoris, purified the complexes, and compared their functional properties. α 2 β 2 and α 2 β 3 differ significantly from both α 2 β 1 and α 1 β 1 in having a higher K 0.5 K + and lower K 0.5 Na + for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K + and shift of the E 1 P-E 2 P conformational equilibrium toward E 1 P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α 2 β 2 and α 2 β 3 over α 1 β 1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K + affinity of α 2 β 2 could allow an acute response to raised ambient K + concentrations in physiological conditions and explain the importance of α 2 β 2 for cardiac muscle contractility. The high sensitivity of α 2 β 2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α 2 β 2 -selective digoxin derivatives for reducing cardiotoxicity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)